Hyperactivity of Purkinje cell and motor deficits in C9orf72 knockout mice

Abstract

A hexanucleotide (GGGGCC) repeat expansion in the first intron of the C9ORF72 gene is the most frequently reported genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The cerebellum has not traditionally been thought to be involved in the pathogenesis of C9ORF72-associated ALS/FTD, but recent evidence suggested a potential role. C9ORF72 is highly expressed in the cerebellum. Decreased C9ORF72 transcript and protein levels were detected in the postmortem cerebellum, suggesting a loss-of-function effect of C9ORF72 mutation. This study investigated the role of loss of C9ORF72 function using a C9orf72 knockout mouse line. C9orf72 deficiency led to motor impairment in rotarod, beam-walking, paw-print, open-field, and grip-strength tests. Purkinje cells are the sole output neurons in the cerebellum, and we next determined their involvement in the motor phenotypes. We found hyperactivity of Purkinje cells in the C9orf72 knockout mouse accompanied by a significant increase of the large-conductance calcium-activated potassium channel (BK) protein in the cerebellum. The link between BK and Purkinje cell firing was demonstrated by the acute application of the BK activator that increased the firing frequency of the Purkinje cells ex vivo. In vivo chemogenetic activation of Purkinje cells in wild-type mice led to similar motor deficits in rotarod and beam-walking tests. Our results highlight that C9ORF72 loss alters the activity of the Purkinje cell and potentially the pathogenesis of the disease. Manipulating the Purkinje cell firing or cerebellar output may contribute to C9ORF72-associated ALS/FTD treatment.

Keywords: BK channel; C9orf72; Hyperactivity; Motor behavior; Purkinje cell.

FIG 1.

Impaired coordination and balance in C9orf72 KO mice. (A) In the accelerated rotarod test, C9orf72 KO mice showed a significantly decreased latency to fall than WT mice in the male group and when both sexes were combined; however, there was no significant difference in the latency to fall between female WT and C9orf72 KO mice. Male: 9 WT, 11 KO, average age: 113 days, range: 75-156 days. Female: 10 WT, 10 KO, average age: 121 days, range 84-162 days. Total: 19 WT, 21 KO, average age: 117 days, range 75-162 days. (B) In the beam-walking test, C9orf72 KO mice showed a significant increase in slip numbers in the male group and when both sexes were combined. On the other hand, there was no significant difference in slip numbers between female WT and C9orf72 KO mice. Male: 9 WT, 11 KO, average age: 129 days, range 91-172 days. Female: 10 WT, 10 KO, average age: 137 days, range 100-178 days. Total: 19 WT, 21 KO, average age: 133 days, range 91-178 days). (C) In the first trial of the beam-walking test, C9orf72 KO mice performed comparably to WT mice. While in the second trial, C9orf72 KO mice displayed excessive slips compared to WT littermates. When comparing two trials, C9orf72 KO mice didn’t improve in the second trial, but their WT littermates showed significant improvement or motor learning. (D) Male C9orf72 KO mice didn’t have improved performance in the second trial, while the male WT mice displayed improved performance. Gait analysis showed increased stride length in the male C9orf72 KO mice (E) and decreased front base length in the total and female groups (F). Male: 9 WT, 11 KO, average age: 149 days, range 111-192 days. Female: 10 WT, 10 KO, average age: 157 days, range 120-198 days. Total: 19 WT, 21 KO, average age: 153 days, range 111-198 days. Bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIG 2.

Hyperactivity and abnormal grip strength of the C9orf72 KO mice. (A) C9orf72 KO mice displayed increased total distance in the open field test. Male: 9 WT, 11 KO, average age: 137 days, range 99-180 days. Female: 10 WT, 10 KO, average age: 145 days, range 108-186 days. Total: 19 WT, 21 KO, average age: 141 days, range 99-186 days. (B) A significant interaction between genotype and body weight in the grip strength of forelimbs. In WT mice, heavier mice have stronger forelimb strength. However, such a relationship did not exist in C9orf72 KO mice. Bars represent mean ± SEMMale: 9 WT, 11 KO, average age: 183 days, range 145-226 days. Female: 10 WT, 10 KO, average age: 191 days, range 154-232 days. Total: 19 WT, 21 KO, average age: 187 days, range 145-232 days. (C) In a second cohort, the C9orf72 KO male mice showed a significantly decreased forelimb grip strength than WT male littermates. Male: 11 WT, 4 KO, average age: 238 days, range 198-269 days. . *p < 0.05.

FIG 3.

Hyperactivity of Purkinje cells in C9orf72 KO mice. Sample traces show the spontaneous firing of Purkinje cells in cell-attached mode recording (A). Purkinje cells from C9orf72 KO mice had a higher firing rate (B) and an unchanged coefficient of variation (C). 4 WT male mice, 15 cells; 3 KO male mice, 17 cells, average age: 93 days, 87-103 days. Bars represent mean ± SEM. ***p < 0.001.

FIG 4.

Increased BK channel in C9orf72 KO mice. Representative Western blot images (top) and their quantifications (bottom) of the cerebellar tissue samples (6 WT male mice, 7 KO male mice, average age: 256 days, range 215-296 days). C9orf72 KO mice showed a higher level of the cerebellar BK channels (A) than WT mice. On the other hand, C9orf72 KO mice did not significantly differ in SK channels (B). Cell-attached recording of tonic Purkinje cells before and after NS11021 application. There is a significant increase in the firing frequency (C) in the adult WT tonic Purkinje cells. The bar graphs show mean ± standard errors. *p < 0.05, **p < 0.01.

FIG 5.

Generation of Pcp2-cre/hM3Dq mice. (A) Strategies to generate Pcp2-cre/DREADD mice. Floxed DREADD mice were crossed with Pcp2-cre mice to produce Pcp2-cre/DREADD mice. (B) Representative PCR-based genotyping results for Pcp2-cre/hM3Dq mice. Lanes 1 and 3: WT mouse; Lane 2: hM3Dq heterozygous mouse; Lane 4: Pcp2-cre mouse. (C) Representative images of cerebellum sagittal slices from Pcp2-cre/hM3Dq mice with DREADD expression in the Purkinje cells. (D) Chemogenetic manipulation of Purkinje cell activity in vitro. Representative Purkinje cell recording traces from Pcp2-cre/hM3Dq before (top), during, and after CNO bath application (bottom). GC: granule cell; WM: white matter; PC: Purkinje cell; ML: molecular layer. Scale bar represents 100 μm.

FIG 6.

Rotarod and beam-walking deficits in Pcp2-cre/hM3Dq mice after CNO injection. Males: 6 CNO group, 6 saline group, average age: 152 days, range 62-205 days; Female: 2 CNO group, 3 saline group, average age: 137 days, range 62-205 days). Pcp2-cre/hM3Dq mice, after CNO injection, showed decreased latency to fall (A) and displayed excessive slips as they crossed the beam (B) compared to the saline-injected group. 1–6: trial number. Bars represent mean ± SEM. **p < 0.01.